Historically, cells such as erythrocytes and their derivatives have been utilized in diagnostic tests, such as hemagglutination, for many years. British Patent 1244 344 and also U.S. Pat. Nos. 3,714,345 and 3,715,427 to Hirata disclose methods for the preparation of erythrocytes derivatized with pyruvic aldehyde and formaldehyde for use in hemagglutination reactions. Human type A erythrocytes could be coated with antigens such as bovine serum albumin, bacteriophages and gamma-globulins, and used in agglutination reactions. However, the coating process does not control the orientation of coupling of the antigen.
Hydrazine-derivatized materials such as latex particles and chromatography resins, have been coupled to antigens and antibodies. U.S. Pat. No. 4,421,896 to Linneaus C. Dorman describes the preparation of hydrazine-derivatized latex particles for use in latex particle agglutination reactions. Hydrazinolysis of amide groups yielded carboxylic hydrazide groups after about 7 to 8 hours at a temperature of 50 to about 80 degrees C. However, such conditions are much too rigorous for cellular membranes. Additionally, cellular membranes are not typically composed of mostly carboxylic amide groups.
Methods for immobilization of antibodies to solid supports are also known. For example, antibodies can be bound to solid supports by covalent bonds between aldehydes generated on the carbohydrate side chains of the antibody and hydrazide groups on the solid support. The hydrazide affinity supports the binding of immunoglobulins from a variety of species and the immobilized antibodies retain more of their biological activity when compared to similar pore-size supports employing protein non-site directed immobilization chemistry.
Jonathan M. Gershoni et al., in Analytical Biochemistry 146:59-63 (1985) disclosed the preparation of adipic dihydrazide derivatized enzymes. Gershoni et al. did not apply this method to cellular membranes, which are much more sensitive to reaction conditions and reagents than proteins in solution.
In view of the foregoing limitations of prior art, it is an object of the present invention to provide a methodology for the preparation and quantitation of semisolid particles or cells that have been chemically derivatized, resulting in hydrazide functionalities covalently incorporated onto the surface molecules of the semisolid particles or cells, as well as a method for determining the levels of hydrazide groups that are on particles or cells.
Another object of the present invention is to prepare derivatized cells useful in agglutination reactions, permitting the controlled attachment of antigens and antibodies to the cells. Hydrazine derivatized cells permit such controlled attachments. Yet another object of the invention is to produce hydrazine derivatized cells under conditions which maintain the integrity of the cellular membrane.
A further object of this invention is to attach antigens and antibodies to hydrazine derivatized cells without loss of the antibody activity or the blocking of the antigen epitope of interest.
An additional object of the invention is to achieve optimal antibody coupling by incorporation onto stabilized red blood cells of optimum levels of hydrazide functionalities.